Human/simian chimeric hepatitis A virus vaccine

ABSTRACT

The present invention relates, in general, to a substantially pure preparation of the simian hepatitis A viral isolate AGM-27; a substantially pure preparation of the genomic DNA of simian hepatitis A viral isolate AGM-27; a pharmaceutical composition comprising the simian hepatitis A viral isolate AGM-27; a method of preventing hepatitis A in an animal; and a vaccine comprising the simian hepatitis A viral isolate AGM-27.

This is a divisional of application Ser. No. 07/674,852 filed Mar. 26, 1991 now U.S. Pat. No. 5,476,658.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates, in general, to a hepatitis A virus. In particular, the present invention relates to a simian hepatitis A viral isolate AGM-27 as deposited with the American Type Culture Collection (ATCC), 12031 Parklawn Drive, Rockville, Md. on Aug. 24, 1992 and having ATCC accession number VR2380.

2. Background Information

Hepatitis-A virus (HAV) is a significant human pathogen that is spread via the fecal-oral route. HAV is a picornavirus that is endemic in underdeveloped areas of the world and causes 20-25% of clinical hepatitis in the developed countries.

Hepatitis A virus has been recovered from several species of New or Old World monkeys. It is not clear whether these isolates are true simian strains or human strains that have infected primates in captivity. Human strains can produce acute hepatitis in some higher primates such as the chimpanzee (Dienstag, J. L., et al. (1975) J. Infect. Dis. 132:532-545), marmoset (Saguinus species) (Deinhardt, F., et al. (1975) Amer. J. Med. Sci. 270:73-80) and owl monkey (Aotus trivigatus) (LeDuc, J. W., et al. (1983) Infect. Immun. 40:766-772), but only one group has reported that an Old World monkey (stump-tailed monkey, Macaca speciosa) (Mao, J. S., et al. (1981) J. Infect. Dis. 144:55-60) has developed acute hepatitis after experimental infection. An important question is how different human and simian isolates actually are.

Recently, comparison of partial genome sequences for two simian strains isolated respectively from an owl monkey (Brown, E. A., et al. (1989) J. Virol. 63:4932-4937) and a cynomolgus monkey (Macaca fascicularis) (Balayan, M. S., et al. (1989) FEBS Lett. 247:425-428) revealed that these two simian strains differed greatly from human HAVs in the limited regions examined. The present invention provides the almost complete sequence of a simian HAV obtained from an African green monkey. Extensive sequence differences between this isolate and other simian and human strains are to be noted. In addition, it can be seen that the simian wild-type strain differs from a human wild-type strain in its ability to grow in simian cell cultures.

SUMMARY OF THE INVENTION

It is a general object of this invention to provide a hepatitis A viral isolate.

It is a specific object of this invention to provide the simian hepatitis A viral isolate AGM-27.

It is another object of the invention to provide genomic DNA of the simian hepatitis A viral isolate AGM-27.

It is a further object of the invention to provide a pharmaceutical composition comprising the simian hepatitis A viral isolate AGM-27.

It is another object of the invention to provide a method of preventing hepatitis A in an animal.

It is a further object of the invention to provide a vaccine comprising the simian hepatitis A viral isolate AGM-27.

Further objects and advantages of the present invention will be clear from the description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Scheme of HAV genome and PCR fragments obtained from AGM-27. Nucleotide numbers are based on the genomic map of HM-175 (Cohen, J. I., et al. (1987) J. Virol. 61:50-59). The sizes of PCR fragments shown include the length of oligonucleotide primers.

FIG. 2. Percent identity among HAV nucleotide sequences coding for capsid proteins, calculated by using the programs "GAP" and "DISTANCES" (Devereux, J., et al. (1984) Nucl. Acids. Res. 12:387-395).

FIGS. 3A-3D show some of the important differences between human strain HM-175 and AGM-27. These differences include an extension of the open-reading frame in the translation-initiation region (FIG. 3A); replacement of D-70 by A-70 in the VP3 region (FIG. 3B); replacement of S-102 by A-102 in the VP1 region (FIG. 3C) and a change of the cleavage site from Q-V to Q-T in the VP2-VP1 junction (FIG. 3D).

FIG. 4. Comparison of growth of simian and human HAV in two different cell lines. Equal aliquots of serially diluted samples containing either the cell culture-adapted variant of HM-175 or the wild-type AGM-27 viruses were plated on confluent cell monolayers of CV-1 or FRhK-4 cells. After 20 days incubation, cells were lysed and viral replication was quantified by slot-blot (Ticehurst, J. R., et al. (1987) J. Clin. Microbiol. 25:1822-1829) to determine the titration end point.

FIGS. 5A-5C show transmission of HAV (Strain AGM-27) to primate where FIG. 5A shows transmission to African Green Monkeys, FIG. 5B shows transmission to marmoset monkeys and FIG. 5C shows transmission to chimpanzees.

FIG. 6. Construction of chimeric HAVs between human and simian strain.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a simian hepatitis A viral isolate.

In one embodiment, the present invention relates to a substantially pure preparation of the simian hepatitis A viral isolate AGM-27. The virus was isolated in primary African Green Monkey Cells shown to be free of contaminating viruses and is therefore suitable for oral administration after completion of requisite safety tests. The fact that none of the strains of hepatitis A virus isolated from man until now has characteristics similar to those of AGM-27 and tests in several primate species indicates that this strain will be attenuated for in humans. Therefore, this strain can be expected to be suitable for use as a live vaccine against hepatitis A. Transmission experiments in primates can be used to determine whether this strain can cross-protect against wild type human strains.

In another embodiment, the present invention relates to a substantially pure preparation of the genomic DNA of simian hepatitis A viral isolate AGM-27. In one preferred embodiment, the genomic DNA has the sequence shown in SEQ ID NO: 1.

In a further embodiment, the present invention relates to a pharmaceutical composition comprising the above-described simian hepatitis A viral isolate AGM-27 in an amount effective to prevent hepatitis A in an animal, and a pharmaceutically acceptable diluent, carrier, or excipient. The pharmaceutical composition of the invention includes the viral isolate AGM-27 in a quantity selected depending on the route of administration (preferably, oral (P.O.), subcutaneous, I.M., or I.V.). Appropriate concentrations and dosage unit sizes can be readily determined by one skilled in the art. Suitable amounts might be expected to fall within the range of approximately 10³ to approximately 10⁸ tissue culture infectious doses.

In another embodiment the present invention relates to a method of preventing hepatitis A in an animal comprising administering to the animal the above-described simian hepatitis A viral isolate AGM-27 under conditions such that said hepatitis A is prevented. One skilled in the art will appreciate that the amounts to be administered for any particular treatment protocol can readily be determined.

In a further embodiment, the present invention relates to a vaccine comprising the above-described simian hepatitis A viral isolate AGM-27 in an amount effective to prevent hepatitis A in an animal and a pharmaceutically acceptable diluent, carrier, or excipient. In one preferred embodiment, the animal is selected from the group consisting of humans, apes, and monkeys.

It has been demonstrated that attenuation of one isolate of virulent HAV occurred in parallel with its adaptation to growth in cell culture. Wild-type HAV isolated from clinical materials grows extremely poorly in cell culture: in vivo replication usually cannot be detected in primate cells until 10-12 weeks after inoculation. It is intersecting, therefore, to find that wild type simian HAV grows in primary AGMK cells or the FRhK-4 cell line without prior adaption. While its ability to grow directly in FRhK-4 or AGMK cells distinguished AGM-27 from the wild-type HM-175, its inability to grow in CV-1 cells distinguished it from the cell culture-adapted variant of the human virus.

These structural and biological data demonstrate that the AGM-27 strain differs significantly from any other known strains of HAV and is a good model for further investigation of the molecular basis of HAV cell-culture adaptation and attenuation. The genome of AGM-27 differs from those of human strains in approximately 1400 nucleotide positions while the human wild-type and attenuated strains of HM-175 differ only in 24 nucleotide positions. A live vaccine based on the AGM-27 strain can be expected to be significantly safer in terms of low reversion to a virulent strain. Thus, chimeric viruses between a human attenuated strain and AGM-27 strain can be designed. These chimeras will have structural regions from the human strain in order to have immunogenic properties of human viruses and nonstructural regions from AGM-27 (especially 2B, 2C and 5' end non-coding region; see Emerson, S. U. et al. (1989) In: R. A. Lerner et al (eds) Modern approaches to new vaccines including prevention of AIDS, Cold Spring Harbor Laboratory, pp. 427-430).

The present invention is described in further detail in the following non-limiting examples.

EXAMPLES

The following protocols and experimental details are referenced in the examples that follow:

HAV. The AGM-27 isolate was obtained as a 10% wt/vol liver homogenate in phosphate buffered saline (pH 7.2) from a clinically-ill African green monkey that was naturally infected with hepatitis A virus in a facility for housing primates (Andzhaparidze, A. G., et al. (1987) Vopr. Virus 6:681-686).

Primers. Eighty oligonucleotides, 15 nucleotides in length and previously synthesized for sequencing human HAV HM-175 (Cohen, J. I., et al. (1987) J. Virol. 61:50-59; Cohen, J. I., et al. (1987) Proc. Natl. Acad. Sci. USA 84:2497-2501), were used for PCR and sequencing reactions. Forty-eight new primers, 19-24 nucleotides in length and complementary to plus or minus strands of the AGM-27 genome, were synthesized using an Applied Biosystems 391 DNA Synthesizer.

Preparation of virus RNA template for PCR. One-half ml of 10% liver homogenate from African green monkey 27 (Andzhaparidze, A. G., et al. (1987) Vopr. Virus 6:681-686), 20% SDS (to a final concentration of 1%) and 10 mg/ml proteinase K (to a concentration of 1 mg/ml) were mixed and incubated for 30 min at 55° C. Total nucleic acids were extracted with phenol at 65° C., precipitated by ethanol, and stored in ethanol at -80° C.

Preparation of virus RNA template for PCR of 5' end. Thirty ml of 10% liver homogenate was clarified by centrifugation at 3,000 rpm for 1 h at 4° C. in a Beckman J-6B Centrifuge. The supernatant was centrifuged at 23,000 rpm in a Beckman SW25 rotor for 14 h. The pellet was resuspended in 1 ml of DNase I buffer with 0.1 mg/ml DNase I and incubated at 37° C. for 30 min. RNA was extracted with phenol at 65° C., treated with RNase-free RQI DNase (Promega Biotec, Madison), and centrifuged at room temperature through 5.7 M CsCl, 10 mM EDTA (pH 7.5) at 40,000 rpm for 14 h in a Beckman SW60 rotor. The RNA pellet was dissolved in 200 μl of water, precipitated with ethanol and stored in ethanol at -80° C.

PCR. Five μl of ethanol-precipitated total nucleic acid or 10 μl of CsCl-purified RNA was centrifuged through 500 μl of ethanol, dried, and used for reverse transcription (RT) and PCR. The usual 100 μl RT-PCR mixture contained template, 10 mM Tris-HCl (pH 8.4), 50 mM KCl, 2.5 mM MgCl₂, 0.2 mmol of each dNTP, 1 nmol direct primer, 1 nmol reverse primer, 4 U RNasin (Promega Biotec, Madison), 5 U AMV-RT (Promega Biotec, Madison), 4 U AmpliTaw (Cetus Corp, CA), under 100 μl of light mineral oil. The mixture was incubated 1 h at 42° C., and then amplified by 35 cycles of PCR: 1 min 94° C., 1.5 min at 30-60° C. depending on primers, 1 to 5 min at 72° C. The products of PCR were analyzed on 1% agarose gels.

Antigen-capture PCR method. AGM-27 viruses from 10% liver homogenate were bound by monoclonal antibodies K2-4F2 or K3-4C8 (MacGregor, A., et al. (1983) J. Clin. Microbiol. 18:1237-1243) to 1.5 ml polypropylene microcentrifuge tubes and PCR with primers specific to the VP3 region was performed as described (Jansen, R. W., et al. (1990) Proc. Natl. Acad. Sci. USA 87:2867-2871).

Purification of PCR fragments for sequencing. PCR fragments were cut from 1% agarose gels, purified by using Geneclean (Bio 101, LaJolla, Calif.) and sequenced by using Sequenase (United States Biochemical Corp., Ohio) as described (Winship, P. R., et al. (1989) Nucleic Acids Res. 17:1266).

Computer analysis of sequences. Nucleotide sequences of HAV strains were compared with that of HAV strain AGM-27 by using the global alignment program "GAP" from the "GCG" software package (Devereus, J., et al. (1984) Nucl. Acids. Res. 12:387-395) on a VAX 11/750 computer (Digital Equipment, Landover, Md.). Multiple alignments were made by using the GCG program "PRETTY" TO ensure that sequence gaps introduced by GAP were consistent from strain to strain. Percent sequence identity was calculated by using the output from GAP and the shorter sequence length as the denominator in the GCG program "DISTANCES."

Amino acid sequences were compared by using the global alignment program "SEQAP" from the "IDEAS" software package (Goad, W. B., et al. (1982) Nucleic Acids Res. 10:247-263). Percent identity was calculated from the output of SEQAP by using the shorter sequence length as the denominator. Amino acid sequences representing L or VP4 through VP1 and 3B through 3D of HAV strain AGM-27, HAV strain HM-175, encephalomyocarditis virus (Palmenberg, A. C., et al. (1984) Nucleic Acids Res. 12:2969-2985), poliovirus type 1 Mahoney (19; Racaniello, V. R., et al. (1981) Proc. Natl. Acad. Sci. USA 78:4887-4891), foot-and-mouth-disease virus type A12 (Forss, S. K., et al. (1984) 12:6587-6601), rhinovirus type 2 (Skern, T., et al. (1985) Nucleic Acids Res. 13:2111-2126), and rhinovirus type 14 (Callahan, P. L., et al. (1985) Proc. Natl. Acad. Sci. USA. 82:732-736; Stanway, G. P., et al. (1984) Nucleic Acids Res. 12:7859-7875) were compared by using the program "RELATE" (Dayhoff, M. O. (1978) p. 1-8 In Dayhoff M. O., ed. Atlas of protein sequence and structure, vol. 5 suppl. 3. Washington, D.C.: National Biomedical Research Foundation).

Virus growth in cell cultures. RFhK-4 and CV-1 cell monolayers were incubated with 10-fold serial dilutions of AGM-27 virus (10% liver homogenate) or the HM-175 cell culture-adapted virus. Diluted viruses (0.15 ml) were added to monolayers of CV-1 or FRhK-4 cells, incubated 90 min at 34.5° C. and then 1 ml of DMEM with 10% fetal calf serum was added. After 20 days incubation in a CO₂ incubator, 1 ml of 2× proteinase K buffer was added to each well to solubilize the cells. Each well contained approximately the same number of cells as measured by light microscopy and determination of total DNA in cell lysates. RNA was isolated from an 80 μl aliquot by proteinase K digestion followed by phenol extraction and used for slot-blot hybridization (Ticehurst, J. R., et al. (1987) J. Clin. Microbiol. 25:1822-1829) with [³² P] labeled riboprobe corresponding to the negative strand of the entire HM-175 genome.

Example 1 Comparison of the AGM-27 Genome Sequence With Other HAV Genomes

The primary structure of the AGM-27 genome was determined from overlapping PCR fragments (FIG. 1). These fragments encompassed the genome corresponding to nucleotides 36-7477 of HM-175 (Cohen, J. I., et al. (1987) J. Virol. 61:50-59). Direct sequencing of these fragments provided AGM-27 sequence between nucleotides 59 and 7458.

The extreme 3' end primer effective in PCR was complementary to the region just before the start of poly A. The extreme 5' end primer effective in PCR corresponded to nucleotide 36-53 of the HM-175 sequence. Five other primers to the region of HM-175 5' to nucleotide 36 were not effective in PCR when used with the AGM-27 genome, although 2 of them were highly effective in PCR of HM-175. Therefore, there may be extensive differences between AGM-27 and HM-175 in the 5' terminus of the genome.

Overall, the nucleotide and amino acid sequences of HAV strain AGM-27 are less similar to those of human HAV strains than the human strains are to each other and are no more similar to those of HAV strain PA21, ioslated from an owl monkey, than to other HAV strains (Table I, FIG. 2). Statistical analysis performed with the program RELATE (Dayhoff, M. O. (1978) p. 1-8 In Dayhoff M. O., ed. Atlas of protein sequence and structure, vol. 5 suppl. 3. Washington, D.C.: National Biomedical Research Foundation) indicated that the amino acid sequences of HAV strains AGM-27 and HM-175 representing VP4 through VP1 and 3B through 3D have little similarity to corresponding regions from other picornaviruses; in these regions, HAV strain AGM-27 did not appear to be more similar than HAV strain HM-175 to any particular picornavirus.

In particular, four of the changes noted lead to unique characteristics of the AGM-27 isolate. These important differences between AGM-27 and all other known human and simian HAVs are detailed in FIGS. 3A-3D. One of the four changes (FIG. 3b) leads to substitution of Asp-70 with Ala-70 in VP3. This substitution is present in neutralization-resistant mutants of HM-175 that lack the ability to bind monoclonal antibody K2-4F2 but still can bind monoclonal antibody K3-4C8 (Ping, L-H., et al. (1988) Proc. Natl. Acad. Sci. USA 85:8281-8285). Using the antigen-capture PCR technique, specific PCR product was obtained when antibody K3-4C8 was used for the capture but not when K2-4F2 was used (data not shown). The other three critical changes are located in the translation-initiation region (FIG. 3a), in the VP1 region (FIG. 3c) and in the VP2-VP1 junction (FIG. 3d).

                                      TABLE 1                                      __________________________________________________________________________     Sequence comparisons of HAV strain AGM-27 and other HAV strains.sup.a          Length                  % Identity versus AGM-27                               Genome                                                                             Nucleotides                                                                              Amino acids                                                                              Nucleotides.sup.b                                                                      Amino acids.sup.c                              region                                                                             AGM-27                                                                              others                                                                              AGM-27                                                                              others                                                                              PA21                                                                              human                                                                               PA21                                                                              human                                       __________________________________________________________________________     5'NT                                                                               .sup. 675.sup.d                                                                     726-734                                                                             --   --   -- 82.0-84.3                                                                           -- --                                          1A   81   69   27   23  93 90-94                                                                               96  96-100                                     1B  666  666  222  222  80.9                                                                              81.7-82.6                                                                           97.3                                                                              96.4-97.7                                   1C  738  738  246  246  82.7                                                                              82.0-83.1                                                                           95.5                                                                              96.7-97.2                                   1D  900  882-900                                                                             300  294-300                                                                             77.9                                                                              80.1-81.4                                                                           92.7                                                                              89.3-94.0                                   2A  567  567  189  189  -- 77.1-81.5                                                                           -- 89.9                                        2B  321  321  107  107  -- 80.7-83.2                                                                           -- 92.5                                        2C  1005 1005 335  335  -- 78.3-79.9                                                                           -- 86.9-91.0                                   3A  216  216-222                                                                              72  72-74                                                                               -- 79.2-83.8                                                                           -- 86.1-88.9                                   3B   69   69   23   23  -- 75-80                                                                               -- 95.7                                        3C  660  657  220  217  -- 81.9-83.4                                                                           -- 95.9-96.3                                   3D  1467 1467 489  489  -- 81.5-81.9                                                                           -- 90.4-91.0                                   3'NT                                                                                64  63-64                                                                               --   --   -- 75-89                                                                               -- --                                          __________________________________________________________________________

Example 2 Growth of AGM-27 in Cell Cultures

FRhK-4 and CV-1 cell lines were inoculated with 10-fold dilutions of 10% liver homogenate containing AGM-27. Preliminary experiments had shown that growth of AGM-27 could be detected by day 14 in either primary African green monkey kidney (AGMK) or FRhK-4 cells by immunofluorescence and slot blot assays. Therefore, virus replication after 20 days was assayed by slot blot and radioautography (FIG. 4). The AGM-27 virus grew well, even when diluted as much as 10⁻⁴, in FRhK-4 cells but replication in CV-1 cells was not detected. In comparison, cell culture-adapted HM-175 strain grew relatively well in both cell cultures: growth was detected in both cultures when the inoculum was diluted 10⁻⁶ and in neither culture when the inoculum was diluted 10⁻⁷.

Example 3 Use of AGM-27 as a Vaccine

Cross-protection experiments based on immunization of chimpanzees with AGM-27 virus followed by challenge with human virulent HAV are currently under way. Infection of 2 chimpanzees with AGM-27 (FIG. 5C) has resulted in animals with high titers of anti-HAV antibodies and no sign of disease. In contrast, two African green monkeys (AGM) (FIG. 5A) and two marmoset monkeys (S. mystrx) (FIG. 5B) developed hepatitis when administered the same inoculum parenterally. Therefore, this simian strain behaves similarly to human attenuated strain HM-175 in chimpanzees, but not in marmosets.

Example 4 Construction of Chimeras

Chimeras can be constructed by religation of purified cDNA fragments generated by digestion with restriction enzymes. A chimera HAV has been constructed between human and simian strains using the enzymes Sac I and EcoRI (FIG. 6). Segments will also be exchanged using PCR generated single-stranded DNA as a mutagenic oligonucleotide (Wychowski, C. et al (1990) Nucleic Acids Res. 18:913-918).

    __________________________________________________________________________     #             SEQUENCE LISTING                                                 - (1) GENERAL INFORMATION:                                                     -    (iii) NUMBER OF SEQUENCES: 1                                              - (2) INFORMATION FOR SEQ ID NO: 1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                           #pairs    (A) LENGTH: 7400 base                                                          (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                 #1:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                    - CTTGATACCT CACCGCCGTT TGCCTAGGCT ATAGGCTTCT TCCCTACACC CT - #TGTTTGTT          60                                                                           - TTTTTTTTTT TTTTTTGTGT GTTTGTAAAT ATTAATTCCT GCAGGTTCAG GG - #TTCTTAA          120                                                                           - TTGTTCTGCT ATACAGACAC TCTTTTCACG CTTTCTGTCA TCTTATTTCC TG - #GGCTCTC          180                                                                           - CCTTGCCCAA GGCTCTGGCC GTTGCGCCCG GCGGGGTCAA CTCCATGGTT AG - #CATGGAG          240                                                                           - TGTAGGAGTC TAAATTGGGG ACGCAGATGC TAGGAACGTC GCCCTGCAGT GT - #TAACCTG          300                                                                           - CTTTCATGAA GCTCTTTGAT CTTCTACAAG AGGTAGGCTA CGGGTGAAAC CT - #CTTAGAT          360                                                                           - AATACTCCTA TGGAGAGATA TCTTGAATAG GGTAACAGCG GTGGATATTG GT - #GAGTTCC          420                                                                           - TTGGGACAAA AACCATTCAA CACCGGAGGA CTGACTCTCA TTCAGTAGTT GC - #ATTGAGT          480                                                                           - AATTGTCTGT CAGGGCTGTC TTTGGGTTTA ATTCCTGGCC TCTCTGTGCT TA - #GGGCAAA          540                                                                           - CATTTCCTGG CCTTAAATGG AGTTCTGTGA GAGGGAACTC CTCCTTTATA TG - #CTGGACA          600                                                                           - ATTTTGGGGC CTTAGGGTTA TGGTTTGCCT CTGAGGTACT CAGGGGCATT TA - #GGTTTTT          660                                                                           - CTCATTTATA TGTTTATGAT GATGAATATG TCTAAACAAG GTATTTTCCA GA - #CTGTTGG          720                                                                           - AGTGGCCTTG ACCACATACT GTCTTTAGCA GATGTGGAGG AAGAGCAAAT GA - #TACAGTC          780                                                                           - GTGGACAGGA CAGCTGTCAC TGGTGCTTCT TATTTTACTT CTGTAGACCA AT - #CTTCAGT          840                                                                           - CATACGGCAG AAGTTGGTGC ACATCAGACA GAGCCTCTTA AGACATCAGT AG - #ATAAACC          900                                                                           - GGTTCAAAGA AAACCCAAGG AGAGAAATTT TTCCTAATAC ATTCTGCAGA TT - #GGTTAAC          960                                                                           - ACACATGCTT TGTTTCATGA AGTCGCCAAA TTGGATGTTG TTAGTTTGTT GT - #ACAATG          1020                                                                           - CAATTTGCTG TACAGGGTTT GTTGAGATAC CATACTTATG CTAGATTTGG AA - #TTGAAA          1080                                                                           - CAAGTCCAGA TTAATCCCAC TCCCTTTCAG CAGGGAGGTC TTATTTGTGC AA - #TGGTTC          1140                                                                           - GGAGACCAAG GTTATGGTTC CATAGCCTCA TTGACAGTTT ATCCACATGG TC - #TCTTGA          1200                                                                           - TGCAACATTA ACAATGTTGT TAGAATCAAA GTTCCATTCA TTTATACTAG AG - #GTGCTT          1260                                                                           - CATTTCAAAG ATCCACAGTA TCCAGTCTGG GAGTTAACTA TTCGTGTTTG GT - #CAGAAT          1320                                                                           - AATATAGGAA CTGGTACTTC TGCTTATACA TCATTGAATG TCTTGGCTAG AT - #TCACTG          1380                                                                           - TTAGAGCTTC ATGGATTGAC ACCATTATCT ACACAAATGA TGAGGAATGA AT - #TTAGAG          1440                                                                           - AGTACAACTG AAAATGTGGT TAATTTGTCA AATTACGAGG ATGCTAGAGC AA - #AGATGT          1500                                                                           - TTTGCACTTG ATCAGGAAGA TTGGAAAACA GATCCCTCGC AAGGAGGAGG AA - #TCAAAA          1560                                                                           - ACTCATTTTA CAACATGGAC TTCAATTCCC ACGCTTGCTG CACAGTTTGC AT - #TTAATG          1620                                                                           - TCTGCATCTG TGGGGCAGCA AATTAAGGTG ATCCCTGTTG ATCCTTATTT TT - #ATCAGA          1680                                                                           - ACCAATTCAA ATCCAGACCA AAAGTGTATT ACTGCTTTAG CTTCTGTCTG TC - #AGATGT          1740                                                                           - TGCTTTTGGA GGGGAGATCT TGTTTTTGAT TTTCAGGTTT TCCCCACAAA AT - #ATCACT          1800                                                                           - GGGAGGTTGT TATTTTGTTT TGTGCCAGGG AATGAGTTGA TAGATGTTTC AG - #GTATAA          1860                                                                           - CTGAAGCAGG CAACTACTGC ACCCTGTGCT GTTATGGATA TAACAGGAGT TC - #AGTCAA          1920                                                                           - TTGAGATTTA GAGTGCCTTG GATCTCTGAT ACACCTTACA GAGTGAATAG AT - #ACACAA          1980                                                                           - TCAGCTCACC AGAAAGGAGA GTATACAGCT ATTGGGAAGT TGATTGTTTA TT - #GTTATA          2040                                                                           - AGGCTTACCT CACCCTCAAA TGTTGCTTCC CATGTTAGGG TTAATGTTTA TC - #TTTCTG          2100                                                                           - ATAAATTTGG AATGTTTTGC ACCCCTATAT CATGCAATGG ATGTGACATC AC - #AGACAG          2160                                                                           - GATGATTCAG GTGGGTTTTC AACTACAGTT TCTACAGAAC AGAATGCTCC TG - #ATCCTC          2220                                                                           - GTTGGAATTA CCACTATTAA GGATTTAAAA GGGAAGGCAA ATAGAGGAAA GA - #TGGATG          2280                                                                           - TCTGGCATTC AAGCACCAGT GGGTGCTATT ACAACCATTG AGGATCCAGT GT - #TAGCTA          2340                                                                           - AAAGTTCCTG AGACTTTTCC AGAATTGAGA CCAGGTGAAT CTAGACATAC TT - #CAGATC          2400                                                                           - ATGTCTATTT ACAAATTTAT GGGGAGGTCA CACTTTCTTT GTACATTTAC TT - #TCAATG          2460                                                                           - AACAATAGGG AGTATACTTT TCCAATAACA CTGTCCTCTA CATCGAATCC AC - #CTCATG          2520                                                                           - TTACCATCAA CACTGAGGTG GTTTTTCAAC CTTTTTCAAT TGTATAGAGG GC - #CATTGG          2580                                                                           - TTGACTATTA TAATTACAGG TGCTACTGAT GTGGATGGCA TGGCTTGGTT TA - #CTCCTG          2640                                                                           - GGCCTAGCTG TGGATACTCC CTGGGTTGAA AAGCAATCAG CGTTGACTAT TG - #ATTATA          2700                                                                           - ACTGCTCTTG GGGCTATTAG GTTTAACACT AGGAGAACAG GAAATATTCA GA - #TTAGAC          2760                                                                           - CCTTGGTATT CATACCTTTA TGCTGTTTCT GGCGCTTTGG ATGGACTTGG GG - #ACACTA          2820                                                                           - GATTCGACTT TCGGGTTGGT CTCTATTCAG ATTGCCAATT ATAATCATTC AG - #ATGAAT          2880                                                                           - CTGTCATTCA GTTGTTATCT TTCAGTTACT GAACAATCAG AATTTTATTT TC - #CAAGGG          2940                                                                           - CCTCTCAATT CTAATGCTAT GATGGTTTCT GAGTCCATGC TAGATCGCAT TG - #CAAGTG          3000                                                                           - GATTTAGAAT CATCAGTTGA TGACCCAAGA TCAGCAGAGG ACAAAAGGTT TG - #AAAGTC          3060                                                                           - ATTGAGCAGG GCAAGCCATA CAAAGAATTA AGAATGGAAG TTGGGAAGCA GA - #GATTGA          3120                                                                           - TATGCCATGG AGGAGTTATC AAATGAAATT TTACCACCTC CTCGGAAAGT GA - #AAGGAC          3180                                                                           - TTTTCTCAAG CTAAAATTTC TTTATTTTAT ACAGAAGACC ATGAAATTGT GA - #AGCTTT          3240                                                                           - TGGAAAGGTC TCACAGCTGA TACAAGAGCT CTCAGGAGAT ATGGTTTTTC TC - #TTGCTG          3300                                                                           - GGAAGAAGTG TGTGGACTCT TGAGATGGAA GCTGGAGTTC TGACTGGAAG GA - #TGATCA          3360                                                                           - TTGAATGATG AAAAGTGGAC TGAGATTAAG GATGATAAGA TAGTGGCTTT GG - #TAGAGA          3420                                                                           - TTTACATCTA ATAAGAATTG GTCTAAAGTC AATTTTCCAC ATGGGATGCT AG - #ATTTGG          3480                                                                           - GAGATAGCAT CAAATTCAAA GGATTTTCCT AATATGTCTG AGACTGACTT GT - #GTTTTC          3540                                                                           - TTACATTGGT TGAATCCTAA GAAGATAAAT CTAGCTGATA GAATGCTTGG AT - #TGTCTG          3600                                                                           - GTTCAGGAAA TTAAGGAACA GGGTGTTGGC TTAATAGCTG AATGTAGAAC AT - #TTTTAG          3660                                                                           - TCTATAGCTG GCACTTTGAA ATCAATGATG TTTGGGTTTC ATCAGTCTGT TA - #CTGTGG          3720                                                                           - ATAATTAATA CTGTCTTGTG TTTTGTTAAG AGTGGGATCC TTCTTTATGT TA - #TTCAGC          3780                                                                           - TTGAATCAAA ATGAACACTC TCATATTATA GGGCTTTTAC AGGTGATGAA TT - #ATGCAG          3840                                                                           - ATTGGTTGCT CTGTGATTTC TTGTGGAAAG ATATTCTCAA AAATGTTAGA AA - #CAGTCT          3900                                                                           - AATTGGCAGA TGGATTCTAG AATGATGGCT CTTAGAACAC AGAGTTTCTC TA - #ATTGGT          3960                                                                           - AGAGACATAT GTTCGGGGAT AACCATTTTC AAAAATTTTA AGGATGCTAT TT - #TCTGGC          4020                                                                           - TACACTAAAT TAAAGGATTA TTATGATTCT AACTATGGGA AAAAGAAGGA TG - #TTCTGA          4080                                                                           - GTTTTAAAAG AAAATCAGCA TAGGATTGAG AAAGCCATTG AAGAGGCTGA TC - #AGTTCT          4140                                                                           - GTTTTGCAGA TTCAGGACGT TGAGAAGTCA GAGCAATATC AGAAGGGAGT TG - #AACTCA          4200                                                                           - CAGAAATTGA GAACAGTTCA TTCCCTGGCC CAGGTCGACT CTAGTTTGAT GT - #CTCATT          4260                                                                           - TCACCACTGA GAGATTGTAT TGCTAGAGTC CATCAAAAAC TTAAGAATTT AG - #GCTCAA          4320                                                                           - AATCAGGCTA TGGTGACTAG GTGTGAACCT GTGGTCTGTT ATTTATATGG TA - #AGAGAG          4380                                                                           - GGAGGAAAGA GTTTAACTTC TATTGCATTG GCAACAAAAA TTTGCAAACA TT - #ATGGTG          4440                                                                           - GAACCAGAAA AGAATATATA TACAAAACCT GTTGCTTCAG ACTACTGGGA TG - #GATATA          4500                                                                           - GGTCAATTGG TTTGTATCAT TGATGACATT GGTCAAAATA CTACAGATGA AG - #ATTGGT          4560                                                                           - GATTTTTGTC AATTGGTGTC TGGTTGTCCT ATGAGGTTAA ATATGGCTTC TT - #TGGAAG          4620                                                                           - AAAGGGAGAC ACTTTTCTTC CCCGTTTATA ATTGCCACAT CAAATTGGTC AA - #ATCCAA          4680                                                                           - CCTAAGACTG TTTATGTGAA GGAAGCTATA GATCGCCGCC TTCATTATAA GA - #TTGAAG          4740                                                                           - AAACCAGCAT CTTTTTACAA AAATGCACAC AATGATATGC TCAATGTGAA TC - #TTGCAA          4800                                                                           - AATAATGATG CCATTAAAGA CATGTCCTGT GTAGATTTAC TGATGGATGG CC - #ATACTG          4860                                                                           - TCTTTATCTG AGCTTTTAAA TTCTCTTGTT ATGACAGTTG AAATTAGAAA AC - #AAAATA          4920                                                                           - TCAGAATTTA TGAAATTGTG GTCACAGGGT GTGTCAGATG ATGATAATGA CA - #GTGCAG          4980                                                                           - GCTGAGTTCT TCCAGTCTTT TCCATCAGGA GAACCCTCAA ATTCTAAGTT AT - #CTAGTT          5040                                                                           - TTCAAGGCGG TCACTAATCA TAAGTGGGTT GCTATTGGAG CTGCTGTTGG AG - #TTCTGG          5100                                                                           - GTCTTAGTGG GAGGTTGGTT TGTGTACAAG CATTTTACCA AAGAAGAACC AA - #TACCAA          5160                                                                           - GAAGGAGTGT ATCATGGAGT AACCAAACCT AAACAGGTTA TCAAATTGGA TG - #CTGATC          5220                                                                           - GTTGACTCCC AATCTACTCT TGAGATAGCT GGACTAGTTA GGAAGAATTT GG - #TTCAAT          5280                                                                           - GGAGTTGGGG AGAAGAATGG ATGTGTTAGG TGGGTCATGA ATGCTTTAGG TA - #TTAAAG          5340                                                                           - GATTGGCTGC TGGTCCCCTC ACATGCATAC AAATTTGAGA AAGATTATCA AA - #TGATGG          5400                                                                           - TTTTATTTTA ATAGAGGAGG AACTTATTAT TCAATTTCTG CTGGTAATGT TG - #TAATCC          5460                                                                           - TCTTTGGATG TTGGTTTTCA GGATGTTGTT TTGATGAAGG TTCCTACAAT TC - #CAAAGT          5520                                                                           - AGAGATATAA CTGAGCATTT TATTAAGAAG AATGATGTTC CAAGAGCTTT GA - #ATAGAT          5580                                                                           - GCTACACTTG TTACAACAGT TAATGGGACA CCAATGCTGA TTTCCGAAGG TC - #CACTTA          5640                                                                           - ATGGAAGAAA AGGCCACTTA TGTCCATAAG AGAAATGACG GAACTACTGT TG - #ATTTGA          5700                                                                           - GTTGATCAAG CTTGGAGGGG AAAAGGTGAG GGCCTCCCAG GTATGTGTGG TG - #GAGCTC          5760                                                                           - ATTTCCTCAA ATCAGTCAAT ACAAAATGCC ATTCTTGGGA TTCATGTTGC AG - #GTGGCA          5820                                                                           - TCTATTTTGG TTGCCAAACT TGTGACTCAG GAAATGTTCC AGAACATTGA AC - #AAAAAG          5880                                                                           - ATAGAAAGTC AGAGGATAAT GAAAGTGGAA TTCACTCAGT GTTCAATGAA TG - #TGGTCT          5940                                                                           - AAAACGCTTT TTAAAAAGAG TCCAATTCAT CATCACATTG ATAGGAACAT GA - #TTAATT          6000                                                                           - CCTGCTGTAA TGCCTTTTTC TAAAGCTGAG ATTGATCCTA TGGCTGTTAT GT - #TGTCTA          6060                                                                           - TATTCTCTTC CTATTGTTGA AGAGCCAGAT GATTATAAGA TGGCTTCCAT TT - #ATTTCC          6120                                                                           - AATAAAGTAA TGGGGAAAAC TTTTCTTGTT GATGACTTTT TGGATATAGA TA - #TGGCAA          6180                                                                           - ACAGGTGCTC CAGGAATAGA TGCTATTAAT ATGGATTCTT CACCAGGATT TC - #CTTATG          6240                                                                           - CAGGAGAAGT TGACAAAGAA AGACTTGATC TGGTTGGATG AGAATGGGCT GC - #TGTTAG          6300                                                                           - GTTCATCCAA GGCTTGCTCA AAGAATCTTG TACAACACAG TTATGATGGA GA - #ATTGTT          6360                                                                           - GATCTTGATG TGGTCTTTAC AACATGTCCC AAGGATGAAC TTAGGCCTCT GG - #ACAAAG          6420                                                                           - TTGGAATCAA AGACTAGAGC AATTGATGCT TGTCCATTGG ATTATACAAT TC - #TTTGTA          6480                                                                           - ATTTATTGGG GTCCTGCTAT TAGTTACTTT CAATTGAATC CTGGATTTCA CA - #CAGGAG          6540                                                                           - GCTATTGGAA TTGATCCGGA TAGACATTGG GACGAGTTGT TTAAAACAAT GG - #TTAGAT          6600                                                                           - GGTGATGTAG GTTTAGACCT TGATTTTTCA TCATTTGATG CTAGTCTTAG TC - #CTTTTA          6660                                                                           - ATAAGAGAGG CAGGGAGAAT TTTGAGTGAA ATGTCAGGGA CACCCTCACA CT - #TTGGAG          6720                                                                           - GCCTTGATTA ATACAATCAT TTATTCCAAG CATTTGTTGT ACAATTGTTG TT - #ATCATG          6780                                                                           - TATGGTTCCA TGCCATCAGG GTCCCCTTGT ACAGCACTTT TAAATTCAAT TG - #TAAACA          6840                                                                           - GTTAATTTGT ACTATGTGTT TTCAAAAATT TTTAGGAAGT CTCCTGTTTT CT - #TTGGAG          6900                                                                           - GCTCTGAAGA TTCTTTGTTA TGGAGATGAT GTCCTCATTG TTTTTTCCAG AA - #ATGTCC          6960                                                                           - ATTGATAATT TGGAATCTAT TGGACAGAAA ATTGTAGATG AGTTTGGAAA AT - #TAGGCA          7020                                                                           - ACTGCAACAT CAGCAGACAA GTCTGTTCCT AAGTTGAAAC CTATTTCTGA GC - #TCACTT          7080                                                                           - CTTAAAAGAT CATTCAATCT TGTTGAAGAT CGGATTAGAC CTGCAATTTC AG - #AGAAAA          7140                                                                           - ATTTGGTCTC TCGTTGCTTG GCAGAGAAGC AATGCTGAAT TTGAACAGAA TT - #TGGAAA          7200                                                                           - GCTCAATGGT TTGCTTTTAT GCATGGTTTT GAATTTTATC AGAAATTTTA CC - #ATTTTG          7260                                                                           - CAGTCCTGCC TGGAGAAAGA GATGGTAGAA TACAGATTGA AATCATATGA TT - #GGTGGA          7320                                                                           - ATGAAGTTTT ATGATCAGTG CTTTGTTTGT GACCTCACAT GATTTGTTTA AA - #CAAACC          7380                                                                           #                 740 - #0                                                     __________________________________________________________________________

All publications mentioned hereinabove are hereby incorporated in their entirety by reference.

While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be appreciated by one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention and appended claims. 

What is claimed is:
 1. A hepatitis A virus having a genome consisting of a human attenuated hepatitis A virus sequence wherein the 5' noncoding region has been replaced by the 5' noncoding region of simian hepatitis A virus AGM-27.
 2. A hepatitis A virus having a genome consisting of a human attenuated hepatitis A virus sequence wherein the 2A nonstructural region has been replaced by the 2A nonstructural region of simian hepatitis A virus AGM-27.
 3. A hepatitis A virus having a genome consisting of a human attenuated hepatitis A virus sequence wherein the 2B nonstructural region has been replaced by the 2B nonstructural region of simian hepatitis A virus AGM-27.
 4. A hepatitis A virus having a genome consisting of a human attenuated hepatitis A virus sequence wherein the 2C nonstructural region has been replaced by the 2C nonstructural region of simian hepatitis A virus AGM-27.
 5. A hepatitis A virus having a genome consisting of a human attenuated hepatitis A virus sequence wherein the 2A, 2B and 2C nonstructural regions have been replaced by the 2A, 2B and 2C nonstructural regions of simian hepatitis A virus AGM-27. 